RI detector system appropriate equilibration time

[Deppizzymo]

Hello

We are running an Agilent 1100 series HPLC with an RI detector. My historical experience with HPLCs was with a system that ran 24/7 in a corn syrup refinery that used water as the mobile phase and so we really never had to equilibrate the systems except for maintenance. It has also been so long ago that I can't remember how long we would leave the system alone to equilibrate. On our current system, we are running a 75% acetonitrile mobile phase and so we shut the system off/turn it on whenever we are using it. This is a new for us/used system and we have not worked on it enough to know what to expect for equilibration time. I ran my calibration curve the other day and it was less than stellar as far as baseline stability/reproducing similar results between injections. I am used to being able to reproduce results within ~0.15% all day every day with the Waters system I used at my previous job that ran 24/7 365 days a week but this one has not been able to give me anything I would consider consistent within ~1.5%. Any other tips or things to look at would be helpful

Pump is running 1.4 mL/min
Column @ 35C
Detector @ 40C
Column is a Waters WAT084038 silica amino reverse phase 3.9mm(id)/300mm(length)

[TylerSmith123]

Hi Depizzymo,

I'm not an expert in RI detectors, but I can certainly offer some relatively obvious advice about these detectors. Firstly, RI detectors are based off a reference cell and a sample cell. The reference cell is static, and will always remain in the detector where the source of your sample cell is coming from the column, allowing you to observe the changes as your mobile phase (with analytes) refract differently than the blank/reference cell. Temperature is extremely important for this consistency, and, while some RI detectors have heat-exchangers to warm your mobile phase (going to the sample cell) to the reference-cell's temperature, it is likely insufficient causing your running mobile-phase to be at a different temperature than the reference cell's. Additionally, the reference cell can take hours to heat up to the correct temperature, this also impacts the refractive index but on the reference cell side. Personally, I would set both your column and detector temperatures to be the exact same to avoid this imbalance, and maybe insulate the tubing between the detector and column to ensure as little temperature-loss as possible.

Mobile-phase preparation and age can obviously play a big role in this as well as you are observing the refractive difference between your reference cell and sample, so proper/accurate and timely mobile-phase preparation is key for replicable results (things like organic-solvent evaporation are important here).

I hope someone can expand a little more on this, but this is relatively basic RI detector knowledge and I would recommend taking a class that focuses on these detectors where you will actually learn the ins-and-outs of this detection technique.

I hope that helps somewhat and you get some improvement in your results.

[Deppizzymo]

That is very informative and gives me something to immediately try. It's interesting because at my previous employer we had the detectors set at 45C and the column at 85C and the mobile phase was kept on a warmed hot plate.

[Deppizzymo]

Hi Depizzymo,

I'm not an expert in RI detectors, but I can certainly offer some relatively obvious advice about these detectors. Firstly, RI detectors are based off a reference cell and a sample cell. The reference cell is static, and will always remain in the detector where the source of your sample cell is coming from the column, allowing you to observe the changes as your mobile phase (with analytes) refract differently than the blank/reference cell. Temperature is extremely important for this consistency, and, while some RI detectors have heat-exchangers to warm your mobile phase (going to the sample cell) to the reference-cell's temperature, it is likely insufficient causing your running mobile-phase to be at a different temperature than the reference cell's. Additionally, the reference cell can take hours to heat up to the correct temperature, this also impacts the refractive index but on the reference cell side. Personally, I would set both your column and detector temperatures to be the exact same to avoid this imbalance, and maybe insulate the tubing between the detector and column to ensure as little temperature-loss as possible.

Mobile-phase preparation and age can obviously play a big role in this as well as you are observing the refractive difference between your reference cell and sample, so proper/accurate and timely mobile-phase preparation is key for replicable results (things like organic-solvent evaporation are important here).

I hope someone can expand a little more on this, but this is relatively basic RI detector knowledge and I would recommend taking a class that focuses on these detectors where you will actually learn the ins-and-outs of this detection technique.

I hope that helps somewhat and you get some improvement in your results.

Can you heat an RI detector cell without flow going to it?

[TylerSmith123]

Hi Depizzymo,

Like I said, I don't have much experience with these detectors, but I found a good excerpt from something John Dolan (an expert in LC troubleshooting) has said on the topic: "If you are having a hard time distinguishing the source of a baseline problem between the pump and mobile phase as opposed to a temperature-related problem, turn off the pump (or set the flow to 0 mL/min). This will eliminate the pump or mobile-phase problem. If the baseline problem persists, it is because of changing temperature." from a discussion about baseline issues with RI detectors. Here's the link if you would like to read: https://www.chromatographyonline.com/vi ... problems-0
Now I will preface this by saying some people on forum would not recommend this resource because the authors may have anterior motives (selling instruments/parts) but John Dolan is a well-respected and authoritative figure in LC/GC and you can trust what he says. Give the article a read, it will likely help you better than I can. I hope you see some improvement with your baseline!

At your previous employer, were you using an RI detector as well? Something positive about a 24/7 instrument and RI detection is that you almost never have to wait for the flow-cell or column to heat up because they are constantly on. Perhaps you could include a low-flow overnight method that retains the temperature of both your units (cell and column) so in the morning it is ready to go?

Have a good one!

[Consumer Products Guy]

I've posted this before, and this is what I did with the same equipment as you.

1. I bypassed the multichannel mixing valve using the little Agilent adapter.

2. I set up the instrument the night before and had the mobile phase pump all night through the column and system, then back into the solvent reservoir.

[DR]

Came in to add something but found that you guys have crushed it!
Good job, faith in this site restored.

[uzman]

Hi Deppizzymo ,

You also stated that you get bad repeatibility , so it may be an autosampler issue.
Amino columns have stability issues as well.
You may also want to check the linearity of your RI detector in order to isolate the problem.

[Multidimensional]

Running an RID with a mobile phase that is made up of 75% ACN (25% Water) will require that a clean, "unfouled" column is used, that the room temperature, LC system and RID flow cell is maintained to a tight tolerance, the flow rate is stable AND the mobile phase is degassed (using a clean, fully serviced inline degasser module).
Equilibration will take as long as it takes, literally. You need to monitor and wait until the pump and detector signals are stable. Be sure to flush the reference cell BEFORE starting an analysis. BTW: Premix the mobile phase and run on one channel for the best pump stability.

If you are having reproducibility issues, Column 'fouling' and/or poor quality degassing may be to blame. (1) When you run a column (esp an amino) with the same isocratic mp all the time, w/o stopping to wash the column off now and then, it will fill with retained material. Column washing must be integrated in the application. (2) Your mobile phase requires degassing to maintain proper flow, good detection and minimize noise. Depending on make and model, inline degasser modules require professional service every 3-5 years. Failure to service and clean a degasser results in flow path contamination (and well... no degassing).

Here is a link to a free article with some hints and tips in how to use a RID.
"HPLC Baseline Stabilization Tips for Refractive Index Detectors (RI or RID)"; https://hplctips.blogspot.com/2018/02/h ... s-for.html